Study On The Function And Mechanism Of Ubiquitination Enzyme USP10 Regulating AMPK

Part IThe AMP-activated protein kinase(AMPK) is a key regulator of cell metabolic/energy homeostasis. As a crucial sensor of cellular energy status, AMPK activation triggers compensatory ATP-generating mechanisms while attenuating ATP-consuming processes to restore cellular energy balance. AMPK regulates various metabolic processes,and its signal transduction plays a central role in the metabolic syndrome,such as obesity and type 2 diabetes.AMPK activity is tightly controlled. When intracellular ATP levels decrease during energy stress, AMPK is initially activated through AMP or ADP binding and phosphorylation of a threonine residue(Thr 172) within the activation loop of its kinase domain. This phosphorylation regulation is well established in AMPK activation, but whether other post-translational modification functions in this pathway remains elusive. Here, we identified USP10 as a novel deubiquitinating enzyme that regulates the AMPK phosphorylation and activities. Furthermore, we figured out the underlying mechanism. Our study will significantly advance our understanding of AMPK control in cell metabolism.Our project includes the following 2 sections:USP10 regulates AMPKα ubiquitination and activation. 1) After AICAR treatment, AMPK was activated, and accompanied with a decrease of AMPKα ubiquitination. 2) 3) Through the analysis of a public proteomic database, we found that AMPKα could be potentially ubiquitinated at four potential sites. Knock-down of USP10 did not affect ubiquitination levels of the AMPKα 4KR mutant, indicated that the ubiquitination on these sites are regulated by USP10. 4) Immunoblot analysis showed that knockdown of USP10 decreased AMPK activation and the phosphorylation of AMPK substrates ACC1 and Raptor. USP10 deficiency resulted in significant increase in lipid drop formation. 5) By sequence analysis, we found that there is an optimal AMPK substrate motif around Ser76 in the N-terminal of USP10. By using a pan-AMPK substrate antibody, we found that the phosphorylation of USP10 increased significantly after glucose starvation,suggesting that USP10 might be a substrate of AMPK. To confirm this result, we performed an in vitro kinase assay and found that AMPK specifically phosphorylated USP10 in vitro. 6) To study the physiological significant of the phosphorylation of S76, we reconstituted the WT or the S76 A mutant into the USP10 knockdown cells. Our results suggested that the phosphorylation of USP10 is important for its function in AMPK activationDepletion of Hepatic USP10 Leads to Multiple Metabolic Defects. 1) We employed adenovirus-based CRISPR-Cas9 gene editing technology to generate live-specific USP10 knockout(KO) mice, and confirmed the KO efficiency by T7EN1 gene editing and Western blot analysis. 2) Consistent with our cell-based assays, deletion of the Usp10 gene in the liver resulted in a drastic decrease of phospho-AMPK as well as the phosphorylation of AMPK substrates. Furthermore, USP10 liver-specific knockout mice showed multiple metabolic defects, such as increased hepatic triglyceride and cholesterol contents. Usp10 knockout mice showed increased blood glucose level and decreased glucose infusion rate, indicating USP10 regulates hepatic glucose metabolism.Taken together, we report a key molecular mechanism by which AMPK activation is amplified under energy stress. Ubiquitination on AMPKα inhibits AMPK activation. The deubiquitinase USP10 specifically removes ubiquitination on AMPKα to facilitate AMPKα phosphorylation. Under energy stress, USP10 activity in turn is enhanced through AMPK-mediated phosphorylation of Ser76 of USP10. Thus, USP10 and AMPK form a key feedforward loop ensuring amplification of AMPK activation in response to fluctuation of cellular energy status. Disruption of this feedforward loop leads to improper AMPK activation and multiple metabolic defects.Our research reveals a new function of ubiquitination in AMPK regulation and will contribute to further understand the significance of posttranslational modification. The key role of AMPK places it as an ideal therapeutic target for the treatment of obesity, insulin resistance, type 2 diabetes, metabolic syndromes, and cancer. AMPK activators, such as metformin had potential for treatment of insulin resistance and type 2 diabete. Given the important roles in AMPK activation, USP10 could be a potential target for these diseases.Chronic myeloid leukemia(CML) is associated is an uncommon type of blood cancer,which is defined by the chromosomal translocation of the non-specific tyrosine kinase c-ABL(Abelson tyrosine kinase) with BCR(breakpoint cluster region). This fusion gene encodes the chimeric oncogenic fusion protein BCR/ABL, a constitutively active tyrosine kinase.The BCR-ABL kinase inhibitor imatinib is regarded as standard first-line treatment in CML. However, resistance against imatinib can occur, representing an increasing clinical challenge. Currently, the molecular mechanism of BCR/ABL-induced oncogenic effects remain poorly understood. In our previous study, we found that Pololike kinase-1(PLK1) is an important downstream target of c-ABL/ BCR-ABL. c-ABL interacts with and phosphorylates PLK1, leading to cell cycle progress and cell growth. Moreover, PLK1 overexpression is correlated with imatinib resistance in CML patients. Therefore, our study represents a new paradigm for cell cycle control and will provide an effective approach for CML therapy.Our project includes the following 4 sections:c-ABL mediated PLK1 phosphorylation regulated PLK1 stability and activity. The phosphorylation of PLK1 by the ABL kinase inhibits PLK1 ubiquitination and degradation, enhances its activity. 1) The interaction between c-ABL and PLK1 was confirmed by Co-immunoprecipitation and GST-pull down assay in vivo and in vitro. 2) Mapping the c-ABL-PLK1 binding regions revealed that the PBD domain of PLK1 is critical for the interaction with c-ABL, and the SH2/SH3,PTKs domain of c-ABL is required for the binding with PLK1. 3) c-ABL phosphorylated PLK1 by kinase assay in vivo and in vitro. MS analysis of the product from in vitro kinase assay showed that Y217, Y425 and Y445 are candidate phosphorylation sites by c-ABL. 4) Overexpression of c-ABL increased PLK1 protein level, whereas c-ABL knockdown decreased PLK1 protein level. c-ABL significantly prolonged the half-life of PLK1 by cycloheximide treatment. The phosphorylation of PLK1 Y425 by c-ABL inhibited PLK1 ubiquitination and degradation by proteasome inhibitor treatment and ubiquitination assay. 5) c-ABL mediated PLK1 phosphorylation promoted PLK1 activation in mitosis and PLK1 Y425 F inhibited Aurora-A kinase mediated PLK1 Thr 210 phosphorylation in vitro.c-ABL mediated PLK1 phosphorylation regulated cell mitotic entry. 1) c-ABL knockdown cells delayed cell cycle progression by flow cytometry(FACS) analysis. 2) Phosphorylation of PLK1 Y425 regulated G2/M transition by immunofluorescence and FACS analysis. 3) Time lapse assay further confirmed that the phosphorylation of PLK1 Y425 regulates cell mitotic entry.c-ABL-PLK1 axis regulates CML response to chemotherapy. 1) BCR/ABL and PLK1 are highly expressed in primary CML cells, which is determined by Real-time PCR and Western blot. The PLK1 protein level was even higher in cells from imatinibresistant patients than those from imatinib sensitive patients. 2) Imatinib treated CML cells showed decreased the expression of PLK1 on protein level. 3) Overexpression of PLK1 resulted in resistance to imatinib in the K562 cells. 4)combination treatment with imatinib and BI2536 exerted significantly greater in vitro antitumor effects than either agent alone. 5)The combination treatment also extended the survival of CMLbearing mice compared with mice in the control group.c-ABL mediated PLK1 tyrosine phosphorylation correlates with tumor progression and patient survival rate in cervical cancers. 1)Both of the m RNA and protein expression levels of c-ABL and PLK1 were significantly higher in cervical tumor tissue than in normal cervical tissue. Higher levels of PLK1 tyrosine phosphorylation ratio were found in cervical cancer samples. 2)The PLK1 mutant Y425 partially inhibited tumor growth in nude mice. 3)Furthermore, patients with higher PLK1 tyrosine phosphorylation show a poor 5-year survival rate. 4)combination treatment with imatinib and BI2536 exerted significantly greater growth inhibition than either drug alone in cervical cancer cells.In summary, our studies reveal a critical role of the c-Abl-Plk1 axis in mitotic progression and CML response to chemotherapy. c-Abl directly phosphorylates Plk1 and regulates Plk1 stability and activity. Overexpression of Plk1 renders CML patient cells resistant to imatinib treatment. Combination treatment with imatinib and Plk1 inhibitor has a better effect on CML and cervical cancer cells than either drug alone. Our study will provide new insight for the response to imatinib-resistant CML patient therapy. In view of the relationship between PLK1 tyrosine phosphorylation and poor prognosis in cervical cancers, our findings may function as a new prognostic marker and therapy approach for human cervical cancers.